We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
A Mouse Model of Fatigue Induced by Peripheral Irradiation
In This Article
Summary
Abstract
Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.
Introduction
Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments1. The fatigue is neither proportional to recent activity nor alleviated by rest, and it is associated with a wide variety of disturbances related to mood, motivation, attention, and cognition2. The biological causes of CRF are unknown, though it has been shown in many cases to correlate with inflammation and cytokine levels, also in some cases with hemoglobin levels and the function of various hormone systems (see Saligan et al.3 for a review of biological studies of CRF).
Controlled studies using animal models are necessary to understand the behavior and biology associated with this complex condition. While tumor-related4 or chemotherapy-related5,6 fatigue has been studied in rodent models, the etiology of CRF may be treatment-specific. To investigate CRF related to radiation therapy, our group has recently developed a mouse model of irradiation-induced fatigue7. In contrast to existing CRF models involving brain or total body irradiation8,9, this model explores how a change in centrally-driven behavior, like fatigue, can be triggered by a peripherally-targeted irradiation procedure.
The procedure described here is designed to model radiation therapy administered to patients with pelvic cancer, using lead shielding to target the lower abdominal/pelvic region with irradiation. However, by modifying the lead shielding or its placement relative to experimental animals, this procedure could be adapted to model irradiation of other parts of the body. Voluntary wheel-running activity (VWRA) is used to measure fatigue-like behavior; because it's a voluntary and normal behavior10, it should allow the concurrent use of other behavioral and biological tests. We have found that peripheral irradiation is sufficient to reduce VWRA in mice without causing overt morbidity7. Future experiments with this model may help reveal effects of peripheral irradiation on immune and other biological signaling, as well as the downstream changes in the central nervous system that can produce deficits associated with CRF.
Access restricted. Please log in or start a trial to view this content.
Protocol
Ethics Statement: This study was approved by the National Institutes of Health (NIH) Animal Care and Use Committee. All investigators taking part in animal handling and measurement of study outcomes were properly trained by the NIH Office of Animal Care and Use and the National Heart, Lung, and Blood Institute Murine Phenotyping Core. All aspects of animal testing, housing, and environmental conditions used in this study were in compliance with The Guide for the Care and Use of Laboratory Animals11.
1. Housing and Experimental Animals
NOTE: House male C57BL/6 mice (roughly five weeks old upon arrival) individually throughout the experiment and provide ad libitum access to food and water. All cages are kept on a 12:12 h light-dark cycle with the light phase beginning at 6 am and dark phase at 6 pm.
- Identify the mice and assign them to individual standard ventilated mouse cages. Allow 24 h after the identification procedure for recovery.
NOTE: Tail tattoo is recommended as a means of identification to eliminate the possibility that an ear tag could get caught in a running wheel. Tattoo a number on the tail of each mouse, with the number on the tail matching the number written on the mouse's cage. - Allow mice to acclimate to their cages for at least three more days, handling each mouse gently for a period of three minutes per day.
2. Running Wheel Acclimation and Baseline
- Introduce the mice to individual VWRA cages, each equipped with a running wheel connected to an electronic counter for continuous recording.
NOTE: All wheel counters connect to a computer through a single USB interface (see Materials List). The computer software calculates the number of wheel rotations, distance traveled, and average speed across each designated time interval of the specified total duration. Once the recording stops, the data are automatically saved both as text and as spreadsheets. - Initiate recording of VWRA through the computer software interface. Set recording intervals to one hour and the duration to at least five days. Continue recording VWRA for at least five days.
NOTE: At the end of step 2.2, all mice should achieve a relatively consistent amount of daily wheel running activity. If not, then identify and exclude any outliers. - Stop VWRA recording through the software interface, and return the mice to their standard cages described in step 1.1 (cages without running wheels).
- Randomize mice into either sham-irradiated control or irradiated groups.
3. Irradiation
Note: Perform the following steps for all mice in both groups, once per day for three sequential days. Treat mice in the same order each day.
- Anesthetize each mouse by intraperitoneal injection of a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture.
- Confirm anesthesia with a toe pinch and use ointment on the eyes to prevent dryness while under anesthesia.
- Transfer the anesthetized mouse into a lead shielding device. Arrange the mouse in the shielding so that only the lower abdominal/pelvic region is exposed.
NOTE: The shielding is composed of two lead "boxes," with a narrow open space in between that allows radiation exposure to a small, targeted region of the mouse (see Figure 1). - Use medical tape to secure the base of the mouse's tail in position within the shielding.
NOTE: Step 3.4 is optional, but doing so can help ensure that the position of the mouse does not change during the following step. - Transport the shielding device into the irradiator, ensuring that the animal position within the shielding is maintained.
- If the mouse is in the irradiation group, deliver 800 cGy at a dose rate of about 110 cGy/min. If the mouse is in the sham-irradiation control group, leave the mouse in the inactive irradiator for the equivalent time.
NOTE: Optimal irradiator settings will depend on the particular device. The dose rate of 110 cGy/min delivered from a 137Cesium source is the central dose rate of the irradiator used here. The exposure time was adjusted to reach the desired total dose of 800 cGy. - Remove the mouse from the irradiator and shielding, and then return it to its original, standard cage mentioned in step 1.1.
- Continuously monitor the mouse until it has regained sufficient consciousness to maintain sternal recumbency.
4. Radiation-induced Fatigue Measurement
- On the day after completion of three successive days of irradiation, transfer the mice to their individual VWRA cages described in step 2.1.
- Record VWRA, as described in step 2.2, except here setting the recording duration to more than 15 days. At the end of the 15 days, manually stop VWRA recording through the software interface.
NOTE: The data from each recording period are automatically saved as spreadsheets, each of which includes the rotation, distance, and speed measurements for all animals (columns) and at all intervals (rows) throughout the duration of recording. At the end of the experiment, there are two spreadsheets generated by the recording software: one for the pre-irradiation VWRA, and one for the post-irradiation VWRA.
Access restricted. Please log in or start a trial to view this content.
Results
Three batches of mice were run through the protocol described above. There were a total of 16 sham and 20 irradiated (2,400 cGy, 3 x 800 cGy/day) mice. After three consecutive days of irradiation, the irradiated group showed significantly reduced VWRA compared to sham (mixed repeated measures ANOVA: main effect of irradiation treatment, F1,13 = 19.233, p < 0.001). The effect was significant for the first seven days after irradiation (simple main effects, p < 0.05 with Bon...
Access restricted. Please log in or start a trial to view this content.
Discussion
We have described a protocol using targeted peripheral irradiation to induce a reduction of VWRA in mice without confounding morbidity or mortality. Importantly, a simple shielding device allows irradiation in this protocol to target a desired region consistently, mimicking the radiation treatments received by patients with pelvic cancer. In contrast to existing CRF models involving brain or total body irradiation8,9, this model explores how a peripherally-target...
Access restricted. Please log in or start a trial to view this content.
Disclosures
This research was supported by the Division of Intramural Research, National Institute of Nursing Research, National Institutes of Health.
Acknowledgements
The authors would like to thank Michele Allen of the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH) for generously sharing her expertise in murine phenotyping methods and for her ongoing technical assistance, as well as for Timothy Hunt of NHLBI for helping us develop the shielding device. This study is supported by the Division of Intramural Research of the National Institute of Nursing Research of the NIH, and part of the validation trial is supported by a grant from the Oncology Nursing Society Foundation.
Access restricted. Please log in or start a trial to view this content.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| C57BL/6 Mice | Charles River | Strain code 027 (http://www.criver.com/products-services/basic-research/find-a-model/c57bl-6n-mouse) | |
| Ketamine HCl | Putney | 100 mg/mL stock solution | |
| Xylazine HCl | Lloyd Laboratories | 100 mg/mL stock solution | |
| Rodent Tattoo System | AIMS | ATS-3 | http://animalid.com/lab-animal-identification-systems/ats-3-general-rodent-tattoo-system |
| Lead Shielding Apparatus | (custom made) | One-inch thick lead shielding arranged as two boxes with a one-inch thick gap between them for targeted irradiation | |
| Plexiglass shielding container | (custom made) | Plexiglass container filled with styrofoam. Styrofoam cutouts hold the lead shielding in place. | |
| GammaCell 40 Exactor | Best Theratronics | http://www.theratronics.ca/product_gamma40.html | |
| RAD Disk Ultra | Best Theratronics | http://www.theratronics.ca/product_rad.html | |
| Mouse Single Activity Wheel Chamber | Lafayette Instrument Company | #80820 | http://www.lafayetteneuroscience.com/product_detail.asp?itemid=980 |
| Activity Wheel Counter for Computer Monitoring | Lafayette Instrument Company | #86061 | http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1052 |
| Modular Cable for Wheel Counters | Lafayette Instrument Company | #86051-7 | http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1046 |
| USB Computer Interface for Activity Wheel Counters | Lafayette Instrument Company | #86056A | http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1047 |
| Activity Wheel Monitor Software | Lafayette Instrument Company | #86065 | http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1053 |
References
- Minton, O., et al. Cancer-related fatigue and its impact on functioning. Cancer. 119, Suppl 11. 2124-2130 (2013).
- Bower, J. E. Cancer-related fatigue--mechanisms, risk factors, and treatments. Nat Rev Clin Oncol. 11 (10), 597-609 (2014).
- Saligan, L. N., et al. The biology of cancer-related fatigue: a review of the literature. Support Care Cancer. 23 (8), 2461-2478 (2015).
- Norden, D. M., et al. Tumor growth increases neuroinflammation, fatigue and depressive-like behavior prior to alterations in muscle function. Brain Behav Immun. 43, 76-85 (2015).
- Ray, M. A., Trammell, R. A., Verhulst, S., Ran, S., Toth, L. A. Development of a mouse model for assessing fatigue during chemotherapy. Comp Med. 61 (2), 119-130 (2011).
- Zombeck, J. A., Fey, E. G., Lyng, G. D., Sonis, S. T. A clinically translatable mouse model for chemotherapy-related fatigue. Comp Med. 63 (6), 491-497 (2013).
- Renner, M., et al. A murine model of peripheral irradiation-induced fatigue. Behav Brain Res. 307, 218-226 (2016).
- Van der Meeren, A., Lebaron-Jacobs, L. Behavioural consequences of an 8 Gy total body irradiation in mice: Regulation by interleukin-4. Canadian Journal of Physiology and Pharmacology. 79 (2), 140-143 (2001).
- York, J. M., et al. The biobehavioral and neuroimmune impact of low-dose ionizing radiation. Brain Behav Immun. 26 (2), 218-227 (2012).
- Meijer, J. H., Robbers, Y. Wheel running in the wild. Proc Biol Sci. 281 (1786), (2014).
- The Guide for the Care and Use of Laboratory Animals. , 8th Edition, National Research Council of the National Academies. (2011).
- Heredia, L., Torrente, M., Domingo, J. L., Colomina, M. T. Individual housing and handling procedures modify anxiety levels of Tg2576 mice assessed in the zero maze test. Physiol Behav. 107 (2), 187-191 (2012).
- Varty, G. B., Paulus, M. P., Braff, D. L., Geyer, M. A. Environmental enrichment and isolation rearing in the rat: effects on locomotor behavior and startle response plasticity. Biol Psychiatry. 47 (10), 864-873 (2000).
- Pham, T. M., Brene, S., Baumans, V. Behavioral assessment of intermittent wheel running and individual housing in mice in the laboratory. J Appl Anim Welf Sci. 8 (3), 157-173 (2005).
- Knab, A. M., et al. Repeatability of exercise behaviors in mice. Physiol Behav. 98 (4), 433-440 (2009).
- Novak, C. M., Burghardt, P. R., Levine, J. A. The use of a running wheel to measure activity in rodents: relationship to energy balance, general activity, and reward. Neurosci Biobehav Rev. 36 (3), 1001-1014 (2012).
- Mineur, Y. S., Belzung, C., Crusio, W. E. Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behav Brain Res. 175 (1), 43-50 (2006).
- Perhach, J. L. Jr, Barry, H. 3rd Stress responses of rats to acute body or neck restraint. Physiol Behav. 5 (4), 443-448 (1970).
- Iwakawa, M., et al. Different radiation susceptibility among five strains of mice detected by a skin reaction. J Radiat Res. 44 (1), 7-13 (2003).
- Travis, E. L., Peters, L. J., McNeill, J., Thames, H. D. Jr, Karolis, C. Effect of dose-rate on total body irradiation: lethality and pathologic findings. Radiother Oncol. 4 (4), 341-351 (1985).
- Duran-Struuck, R., Dysko, R. C. Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies. J Am Assoc Lab Anim Sci. 48 (1), 11-22 (2009).
- Duran-Struuck, R., et al. Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies. J Transl Med. 6, 10(2008).
Access restricted. Please log in or start a trial to view this content.
Reprints and Permissions
